This proposal describes a five year training plan for the development of an independent translational academic cancer research program. The candidate has had research training as a graduate student and residency training in internal medicine, has completing fellowship training in hematology/oncology, and continues as faculty in a mentored research environment. The proposed program will promote the candidate's further training in advanced cancer biology, mouse models of cancer, and translational breast cancer research. The candidate's mentor is a recognized leader in breast cancer basic science and translational research, and is committed to supervising the PI's research training and fostering his transition to independence. Training will occur in an environment of active clinical practice and basic science investigation in breast cancer, coupled with outstanding resources for the proposed research and with institutional support for the nurturing and development of physician-scientists. The proposed research will focus on understanding molecular mechanisms of resistance to HER2 inhibitors in breast cancer. Amplification of the HER2 oncogene occurs in 25% of human breast cancers. Trastuzumab, an antibody against HER2, and lapatinib, a tyrosine kinase inhibitor of HER2 and EGFR, are effective therapies for patients with HER2+ breast cancer, but most patients treated with these agents eventually relapse, suggesting that tumors acquire or intrinsically possess mechanisms for escape from HER2 inhibition. Aberrant activation of the phosphoinositide-3-kinase (PI3K)-Akt axis is emerging as an important mechanism of resistance to anti-HER2 therapies, supported by preliminary data from cell line model systems of lapatinib resistance. We hypothesize that as a result of the selective pressure of treatment, resistance to HER2 inhibition results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling and/or mutations in the PI3K pathway. Inhibition of these alternate pathways should provide therapeutic targets to prevent or overcome resistance to anti-HER2 treatment. Specific aims are 1) To determine the molecular mechanisms of aberrant PI3K-Akt activation in lapatinib-resistant HER2-overexpressing breast cancer cells; 2) To determine the mechanisms whereby PI3K mutations contribute to PI3K-Akt pathway activation in drug-resistant cells; and 3) To determine whether therapeutic inhibitors of PI3K restore sensitivity to HER2 inhibitors and whether PI3K pathway activation predicts clinical response to HER2 inhibitors. Experiments in Aim 1 will use cell line models of lapatinib resistance to determine the role of HER3 in activating PI3K, or to identify other candidate activators of the PI3K pathway. Several approaches including RNAi knockdown of candidate proteins, immunoprecipitation of PI3K subunits to identify binding partners, and pharmacologic manipulation of suspected resistance pathways will be tested. Aim 2 will focus on understanding the role of PI3K mutations by using mass spectrometry to quantitate mutant PI3K isoforms in resistant cells, and by knockdown of PI3K subunits to test the role of mutations on PI3K signaling. Using xenograft mouse models, Aim 3 will test whether PI3K mutations confer resistance in vivo, and whether combination of PI3K inhibition with HER2 inhbitors provides a strategy to overcome HER2 inhibitor resistance. Further, the hypothesis that activation of the PI3K pathway by PIK3CA mutation or PTEN loss predicts for clinical resistance to HER2 inhibitors will be tested in tumor samples from a neoajuvant clinical trial of lapatinib.